Longitudinal changes in choline concentration and associated factors in human breast milk.

BACKGROUND: Human breast milk is the primary source of choline and choline-containing compounds for infants at early stages of life. Choline data across lactation in Chinese human milk were limited. OBJECTIVE: This study aimed to quantify the five choline compounds in Chinese human breast milk and explore associated factors. METHODS: A total of 540 lactating mothers from the MUAI (Maternal Nutrition and Infant Investigation) study were included. The content of water-soluble choline (free choline, phosphocholine, glycerophosphocholine) and lipid-soluble choline (phosphatidylcholine, sphingomyelin) in 892 human milk samples collected from 0 to 400 days postpartum were examined, and associated factors were explored. RESULTS: Choline concentrations in human milk varied from postpartum day 0-400 (92.06 ± 65.22 to 171.01 ± 47.84 mg/L). Water-soluble choline was the major component (88.6%-93.8%) in human milk and ranged from 793.03 (659.22) to 1544.43 (443.32) µmol/L. Its trajectory followed that of total choline, increasing from colostrum to transitional milk and then declining in mature milk. In contrast, lipid-soluble choline accounted for 6.2%-11.4% over lactation and had an opposite trajectory. Choline composition varied by delivery mode and parity history. CONCLUSION: The concentrations of individual choline and choline-containing compounds during lactation in Chinese human breast milk were described for the first time. Our results address gaps in extant Chinese human milk choline data and support tailored dietary reference intakes for Chinese lactating women and infants. Our data describes the level and profile of choline from 0 to 400 days postpartum in Chinese human breast milk. This is the most updated data on choline and also the first report of water-soluble choline as the predominant type in Chinese human milk. Our results compensate for the deficiencies in data on choline in Chinese human milk. CLINICAL TRIAL REGISTRATION: Clinical Trial Registry number: ChiCTR1800015387. Web link to study on registry: https://www.chictr.org.cn/index.aspx.

Improved Spatial Resolution of Metabolites in Tissue Biopsies Using High-Resolution Magic-Angle-Spinning Slice Localization NMR Spectroscopy.

High-resolution magic-angle-spinning 1H NMR spectroscopy (HR-MAS NMR) is a well-established technique for assessing the biochemical composition of intact tissue samples. In this study, we utilized a method based on HR-MAS NMR spectroscopy with slice localization (SLS) to achieve spatial resolution of metabolites. The obtained 7 slice spectra from each of the model samples (i.e., chicken thigh muscle with skin and murine renal biopsy including medulla (M) and cortex (C)) showed distinct metabolite compositions. Furthermore, we analyzed previously acquired 1H HR-MAS NMR spectra of separated cortex and medulla samples using multivariate statistical methods. Concentrations of glycerophosphocholine (GPC) were found to be significantly higher in the renal medulla compared to the cortex. Using GPC as a biomarker, we identified the tissue slices that were predominantly the cortex or medulla. This study demonstrates that HR-MAS SLS combined with multivariate statistics has the potential for identifying tissue heterogeneity and detailed biochemical characterization of complex tissue samples.

In vivo Metabolic Profiles as Determined by 31P and short TE 1H MR-Spectroscopy : No Difference Between Patients with IDH Wildtype and IDH Mutant Gliomas.

PURPOSE: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1H-decoupled 31P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas. METHODS: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1H decoupled 31P MRSI with 3D CSI (3D 31P CSI), 2D 1H CSI and a 1H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites. RESULTS: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pHi) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1H CSI data for tumor and control tissue showed the anticipated results. CONCLUSION: Using 3D-CSI data acquisition, in vivo 31P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.

Rapid Detection of Small Molecule Metabolites in Serum of Hepatocellular Carcinoma Patients Using Ultrafast Liquid Chromatography-Ion Trap-Time of Flight Tandem Mass Spectrometry.

A method was developed for analyzing broad spectrum small molecule metabolites in the serum of hepatocellular carcinoma (HCC) patients based on ultrafast liquid chromatography-ion trap-time of flight tandem mass spectrometry (UFLC-IT-TOF MS). Serum samples were collected from 80 HCC patients and healthy persons. After pretreatment process for protein precipitation, the supernatant was analyzed with the UFLC-IT-TOF MS to obtain information on the metabonomics of small molecules. The eight compounds of glycocholic acid, choline glycerophosphate, acetyl-L-phenylalanine, oleamide, tetradecanamide, acetylcarnitine, lysolecithin and glycochenodeoxycholic acid in the HCC group were identified with significant differences from those in the health group (P <0.01). By using multidimensional analysis of variation coefficient and principal component analysis for the repeatability and 48 h stability, the method was demonstrated to have good repeatability, excellent precision, and high stability, which can satisfy the metabonomics research requirement. The high throughput and practical usability of the method further shows perspective for metabonomic analysis of large-batch serum samples.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTION: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. WHAT IS KNOWN ALREADY: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm. MAIN RESULTS AND THE ROLE OF CHANCE: DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. LIMITATIONS, REASONS FOR CAUTION: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. WIDER IMPLICATIONS OF THE FINDINGS: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.

Electron-induced dissociation (EID) for structure characterization of glycerophosphatidylcholine: determination of double-bond positions and localization of acyl chains.

Glycerophospholipids are a highly abundant and diverse collection of biologically relevant lipids, and distinction between isomeric and isobaric species is a fundamental aspect for confident identification. The ability to confidently assign a unique structure to a glycerophospholipid of interest is dependent on determining the number and location of the points of unsaturation and assignment of acyl chain position. The use of high-energy electrons (>20 eV) to induce gas-phase dissociation of intact precursor ions results in diagnostic product ions for localizing double-bond positions and determining acyl chain assignment. We describe a high-resolution, tandem mass spectrometry method for structure characterization of glycerophospholipids using electron-induced dissociation (EID). Furthermore, the inclusion of nomenclature to systematically assign bond cleavage sites with acyl chain position and double-bond location enables a uniform platform for lipid identification. The EID methodology detailed here combines novel application of an electron-based dissociation technique with high-resolution mass spectrometry that facilitates a new experimental approach for lipid biomarker discovery and validation.

EVALUATION OF ANTIVIRAL THERAPY TREATMENT FOR LIVER CIRRHOSIS CAUSED BY CHRONIC HEPATITIS C AND HEPATITIS C BY 31P-MRS, BASED ON METABOLITE DETECTION.

This study discusses the application of magnetic resonance spectrum (MRS) to evaluate the efficacy of antiviral therapy in the treatment of liver cirrhosis caused by chronic hepatitis C and hepatitis C, based on metabolite detection. A total of 54 patients with liver cirrhosis caused by chronic hepatitis C and hepatitis C were selected and divided into treatment group and control group. 31P-MRS imaging was carried out on patients in the two groups both before receiving antiviral treatment and 6 months after treatment to compare the change of metabolite ratio (PE+PC)/(GPE+GPC). It was revealed that no statistically significant difference was found in the comparison of (PC+PE)/(GPC+GPE) ratio in the two groups before treatment, but the difference was found 6 months after treatment; ratio of (PC+PE)/ (GPC+GPE) in the treatment group distinctly decreased 6 months after treatment compared to before treatment, with a statistically significant difference, while the control group had no remarkable change or statistical significance. Moreover, 32 patients were found with sustained virus response to antiviral therapy. Of these, 25 patients possessed a decreased ratio of (PC+PE)/ (GPC+GPE), 4 remained without change and 3 had a slightly increased ratio after antiviral treatment. Of 12 patients with no response, 1 had a decreased ratio of (PC+PE)/ (GPC+GPE), 2 remained without change and 9 had a slightly increased ratio. The differences were all statistically significant in comparison of the two groups. 31P-MRS is thought to be effective for evaluating the efficacy of antiviral therapy through non-invasive detection of liver energy metabolism.

Bacterial colonization due to increased nurse workload in an intensive care unit / Colonização bacteriana por causa do aumento da carga de trabalho da equipe de enfermagem em unidade de terapia intensiva / Colonización bacteriana debido al aumento de la carga de trabajo del equipo de enfermería en una unidad de cuidados intensivos

INTRODUCTION: The rates of multiresistant bacteria colonization or infection (MRB+) development in intensive care units are very high. The aim of this study was to determine the possible association between the risk of development of nosocomial infections and increased daily nurse workload due to understaffing in intensive care unit. METHODS: We included 168 patients. Intensity of workload and applied procedures to patients were scored with the Project de Recherché en Nursing and the Omega scores, respectively. The criteria used for infections were those defined by the Centers for Disease Control. RESULTS: Of the 168 patients, 91 (54.2%) were female and 77 (45.8%) were male patients. The mean age of female and male was 64.9 ± 6.2 years and 63.1 ± 11.9 years, respectively. The mean duration of hospitalization in intensive care unit was 18.4 ± 6.1 days. Multiresistant bacteria were isolated from cultures of 39 (23.2%) patients. The development of MRB+ infection was correlated with length of stay, Omega 1, Omega 2, Omega 3, Total Omega, daily PRN, and Total PRN (p < 0.05). There was no correlation between development of MRB+ infection with gender, age and APACHE-II scores (p > 0.05). CONCLUSION: The risk of nosocomial infection development in an intensive care unit is directly correlated with increased nurse workload, applied intervention, and length of stay. Understaffing in the intensive care unit is an important health problem that especially affects care-needing patients. Nosocomial infection development has laid a heavy burden on the economy of many countries. To control nosocomial infection development in the intensive care unit, nurse workload, staffing level, and working conditions must be arranged. .

INTRODUÇÃO: As taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes (BMR+) em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem devido à insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: Cento e sessenta e oito pacientes foram incluídos. O volume da carga de trabalho e os procedimentos realizados em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os definidos pelos Centros de Controle de Doenças. RESULTADOS: Dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do sexo masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR+ foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR+ e gênero, idade e escores no APACHE-II (p > 0,05). CONCLUSÃO: O risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga ...

INTRODUÇÃO: as taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes [BMR (+)] em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem por causa da insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: foram incluídos 168 pacientes. O volume da carga de trabalho e os procedimentos feitos em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os estabelecidos pelos Centros de Controle de Doenças. RESULTADOS: dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR (+) foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR (+) e gênero, idade e escores no Apache-II (p > 0,05). CONCLUSÃO: o risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga de trabalho ...

INTRODUCCIÓN: Las tasas de desarrollo de infección o colonización por bacterias multirresistentes en unidades de cuidados intensivos son muy elevadas. El objetivo de este estudio fue determinar la posible asociación entre el riesgo de desarrollo de infecciones hospitalarias y el aumento de la carga de trabajo diaria del equipo de enfermería debido a la falta de personal en la unidad de cuidados intensivos. MÉTODOS: Ciento sesenta y ocho pacientes fueron incluidos. El volumen de la carga de trabajo y los procedimientos realizados en pacientes fueron evaluados con el uso de instrumentos de medidas como el Proyecto de Investigación en Enfermería (Project de Recherché en Nursing) y el Omega, respectivamente. Los criterios usados para definir infecciones fueron los definidos por los Centros de Control de Enfermedades. RESULTADOS: De los 168 pacientes, 91 (54,2%) eran del sexo femenino y 77 (45,8%) del sexo masculino. La edad media de las mujeres y de los hombres fueron 64,9 ± 6,2 y 63,1 ± 11,9 años, respectivamente. El tiempo medio de ingreso en la unidad de cuidados intensivos fue de 18,4 ± 6,1 días. Las bacterias multirresistentes fueron aisladas a partir de cultivos de 39 (23,2%) pacientes. El desarrollo de infección por bacterias multirresistentes fue correlacionado con el tiempo de ingreso, Omega 1, Omega 2, Omega 3, Omega total, PPE diario y PPE total (p < 0,05). No hubo correlación entre el desarrollo de la infección por bacterias multirresistentes y el sexo, la edad y las puntuaciones en el APACHE-II (p > 0,05). CONCLUSIÓN: El riesgo de desarrollo de infección hospitalaria en una unidad de cuidados intensivos está directamente relacionado con el aumento de la carga de trabajo de enfermería, las intervenciones practicadas y el tiempo de ingreso. La falta de personal en la unidad de cuidados intensivos es un problema de sanidad importante que afecta principalmente a los pacientes que necesitan esos cuidados. La infección hospitalaria ...

Evidence for an N-methyl transfer reaction in phosphatidylcholines with a terminal aldehyde during negative electrospray ionization tandem mass spectrometry.

Lipidomic analysis of the complex mixture of lipids isolated from biological systems can be a challenging process that often involves tandem mass spectrometry and interpretation of both precursor ions and product ions relative to the molecular structure of the lipids. Therefore, detailed understanding of the gas-phase ion chemistry occurring for each class of phospholipids is critically important for an accurate assignment of lipid structure. Some oxidized phosphatidylcholines are known to be biologically active and responsible for pathological events, and are therefore important targets for detection in lipidomic studies. Modification of fatty acyl chains by oxidation may, however, change the behavior of ion formation and decomposition in the mass spectrometer. In this study, we report on the mass-spectrometric behavior of 1-palmitoyl-2-(9'-oxononanoyl)-sn-glycero-3-phosphocholine, a bioactive product of phosphatidylcholine oxidation. In addition to [M-15](-) and the acetate adduct [M+59](-), three additional adduct ions, including [M-H](-), were present in significant abundance in the negative ion electrospray mass spectrum. It was found that this unexpected [M-H](-) ion was formed by the transfer of a methyl group from the choline residue on the polar head group to the aldehyde functionality of the sn-2 substituent, resulting in a 14-Da increase in the mass of the resulting sn-2 carboxylate anion formed by collisional activation of this ion. These results suggest additional rules for understanding the gas-phase ion chemistry of aldehydic phosphatidylcholine molecular species.

Muscle MRS detects elevated PDE/ATP ratios prior to fatty infiltration in Becker muscular dystrophy.

Becker muscular dystrophy (BMD) is characterized by progressive muscle weakness. Muscles show structural changes (fatty infiltration, fibrosis) and metabolic changes, both of which can be assessed using MRI and MRS. It is unknown at what stage of the disease process metabolic changes arise and how this might vary for different metabolites. In this study we assessed metabolic changes in skeletal muscles of Becker patients, both with and without fatty infiltration, quantified via Dixon MRI and (31) P MRS. MRI and (31) P MRS scans were obtained from 25 Becker patients and 14 healthy controls using a 7 T MR scanner. Five lower-leg muscles were individually assessed for fat and muscle metabolite levels. In the peroneus, soleus and anterior tibialis muscles with non-increased fat levels, PDE/ATP ratios were higher (P < 0.02) compared with controls, whereas in all muscles with increased fat levels PDE/ATP ratios were higher compared with healthy controls (P ≤ 0.05). The Pi /ATP ratio in the peroneus muscles was higher in muscles with increased fat fractions (P = 0.005), and the PCr/ATP ratio was lower in the anterior tibialis muscles with increased fat fractions (P = 0.005). There were no other significant changes in metabolites, but an increase in tissue pH was found in all muscles of the total group of BMD patients in comparison with healthy controls (P < 0.05). These findings suggest that (31) P MRS can be used to detect early changes in individual muscles of BMD patients, which are present before the onset of fatty infiltration.

Increased sensitivity of 31P MRSI using direct detection integrated with multi-echo polarization transfer (DIMEPT).

Here, we show that the sensitivity of (31)P MRSI of (31)P spins J-coupled to protons can be increased by almost a factor of three when compared with an optimal direct detection free induction decay. By direct detection integrated with multi-echo polarization transfer (DIMEPT), multiple signals from polarization transfer and direct detection can be acquired in one repetition time, with minimal mutual interference, provided that the number of refocusing pulses in the multi-echo polarization transfer part is even. The DIMEPT sequence was implemented on a 7-T body scanner and tested on a phantom and on the breasts of five healthy volunteers. The in vivo signal-to-noise ratio (SNR) enhancement for the J-coupled phosphomonoesters was 270% when compared with an Ernst angle pulse-acquire sequence. However, the phosphodiester signals, presumably mainly mobile phospholipids, had T2 values that were too short to be enhanced. Uncoupled (31)P spins, with sufficiently long T2 values, such as inorganic phosphate, were SNR enhanced by a factor of 1.9 relative to an Ernst-angle excitation pulse-acquire sequence by multi-echo direct detection.

An in vivo 1H magnetic resonance spectroscopy study of the deep cerebellar nuclei in children with fetal alcohol spectrum disorders.

BACKGROUND: Prenatal alcohol exposure has been linked to impairment in cerebellar structure and function, including eyeblink conditioning. The deep cerebellar nuclei, which play a critical role in cerebellar-mediated learning, receive extensive inputs from brain stem and cerebellar cortex and provide the point of origin for most of the output fibers to other regions of the brain. We used in vivo (1) H magnetic resonance spectroscopy (MRS) to examine effects of prenatal alcohol exposure on neurochemistry in this important cerebellar region. METHODS: MRS data from the deep cerebellar nuclei were acquired from 37 children with heavy prenatal alcohol exposure and 17 non- or minimally exposed controls from the Cape Coloured (mixed ancestry) community in Cape Town, South Africa. RESULTS: Increased maternal alcohol consumption around time of conception was associated with lower N-Acetylaspartate (NAA) levels in the deep nuclei (r = -0.33, p < 0.05). Higher levels of alcohol consumption during pregnancy were related to lower levels of the choline-containing metabolites (r = -0.37, p < 0.01), glycerophosphocholine plus phosphocholine (Cho). Alcohol consumption levels both at conception (r = 0.35, p < 0.01) and during pregnancy (r = 0.38, p < 0.01) were related to higher levels of glutamate plus glutamine (Glx). All these effects continued to be significant after controlling for potential confounders. CONCLUSIONS: The lower NAA levels seen in relation to prenatal alcohol exposure may reflect impaired neuronal integrity in the deep cerebellar nuclei. Our finding of lower Cho points to disrupted Cho metabolism of membrane phospholipids, reflecting altered neuropil development with potentially reduced content of dendrites and synapses. The alcohol-related alterations in Glx may suggest a disruption of the glutamate-glutamine cycling involved in glutamatergic excitatory neurotransmission.

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Thalamic metabolic abnormalities in patients with Huntington's disease measured by magnetic resonance spectroscopy.

Huntington's disease (HD) is a neurologic disorder that is not completely understood; its fundamental physiological mechanisms and chemical effects remain somewhat unclear. Among these uncertainties, we can highlight information about the concentrations of brain metabolites, which have been widely discussed. Concentration differences in affected, compared to healthy, individuals could lead to the development of useful tools for evaluating the progression of disease, or to the advance of investigations of different/alternative treatments. The aim of this study was to compare the thalamic concentration of metabolites in HD patients and healthy individuals using magnetic resonance spectroscopy. We used a 2.0-Tesla magnetic field, repetition time of 1500 ms, and echo time of 135 ms. Spectra from 40 adult HD patients and 26 control subjects were compared. Quantitative analysis was performed using the LCModel method. There were statistically significant differences between HD patients and controls in the concentrations of N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG; t-test, P<0.001), and glycerophosphocholine+phosphocholine (GPC+PCh; t-test, P=0.001) relative to creatine+phosphocreatine (Cr+PCr). The NAA+NAAG/Cr+PCr ratio was decreased by 9% and GPC+PCh/Cr+PCr increased by 17% in patients compared with controls. There were no correlations between the concentration ratios and clinical features. Although these results could be caused by T1 and T2 changes, rather than variations in metabolite concentrations given the short repetition time and long echo time values used, our findings point to thalamic dysfunction, corroborating prior evidence.

Thalamic metabolic abnormalities in patients with Huntington's disease measured by magnetic resonance spectroscopy

Huntington's disease (HD) is a neurologic disorder that is not completely understood; its fundamental physiological mechanisms and chemical effects remain somewhat unclear. Among these uncertainties, we can highlight information about the concentrations of brain metabolites, which have been widely discussed. Concentration differences in affected, compared to healthy, individuals could lead to the development of useful tools for evaluating the progression of disease, or to the advance of investigations of different/alternative treatments. The aim of this study was to compare the thalamic concentration of metabolites in HD patients and healthy individuals using magnetic resonance spectroscopy. We used a 2.0-Tesla magnetic field, repetition time of 1500 ms, and echo time of 135 ms. Spectra from 40 adult HD patients and 26 control subjects were compared. Quantitative analysis was performed using the LCModel method. There were statistically significant differences between HD patients and controls in the concentrations of N-acetylaspartate+N-acetylaspartylglutamate (NAA+NAAG; t-test, P<0.001), and glycerophosphocholine+phosphocholine (GPC+PCh; t-test, P=0.001) relative to creatine+phosphocreatine (Cr+PCr). The NAA+NAAG/Cr+PCr ratio was decreased by 9% and GPC+PCh/Cr+PCr increased by 17% in patients compared with controls. There were no correlations between the concentration ratios and clinical features. Although these results could be caused by T1 and T2 changes, rather than variations in metabolite concentrations given the short repetition time and long echo time values used, our findings point to thalamic dysfunction, corroborating prior evidence.

Profiling phospholipid elution in reversed-phase LC-MS/MS bioanalytical methods in order to avoid matrix effects.

BACKGROUND: Endogenous phospholipids have a profound matrix effect in bioanalytical LC-MS methods and considerable effort is invested in strategies to minimize their impact either by removal during sample processing or chromatographic separation during the analytical run. The aim of the research presented in this article was to investigate the latter approach, under reversed-phase conditions. RESULTS: The retention of glycerophosphocholines (GPCs) in mobile phases employing acetonitrile demonstrated a complex 'U-shaped' relationship with the percentage of organic. Conversely, in mobile phases employing methanol, the relationship between retention and percentage of organic was entirely predictive and unaffected by changes in the mobile phase pH. The GPC elution profile was also qualitatively equivalent irrespective of the species from which the plasma was derived. CONCLUSION: The predictive nature of GPC retention, under reversed-phase chromatography and with MeOH as organic modifier, is an important finding that should allow for a more streamlined and simplified strategy in the development of bioanalytical assays.

Analytical bias between species caused by matrix effects in quantitative analysis of a small-molecule pharmaceutical candidate in plasma.

BACKGROUND: Suppression or enhancement of MS ionization, particularly evident when electrospray is used as the source of ions, has been widely discussed. METHODS: An assay for a small-molecule pharmaceutical in dog plasma between 1-300 ng/ml was validated with a mean bias across the calibration range of 5.0%. When the calibration sample matrix was substituted for human plasma, the mean bias across the range increased to 29.1%. A study of bias originating as a result of matrix effects, arising from endogenous glycerophosphocholine species, in plasma sources is discussed. CONCLUSION: A simple strategy to assess the potential of any unmitigated matrix effect to bias quantitative analysis by nonequivalent ionization induction or suppression is evaluated.

Rapid NMR method for the quantification of organic compounds in thin stillage.

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

Multimodal mass spectrometric imaging of small molecules reveals distinct spatio-molecular signatures in differentially metastatic breast tumor models.

Phosphocholine (PC) and total choline (tCho) are increased in malignant breast tumors. In this study, we combined magnetic resonance spectroscopic imaging (MRSI), mass spectrometry (MS) imaging, and pathologic assessment of corresponding tumor sections to investigate the localization of choline metabolites and cations in viable versus necrotic tumor regions in the nonmetastatic MCF-7 and the highly metastatic MDA-MB-231 breast cancer xenograft models. In vivo three-dimensional MRSI showed that high tCho levels, consisting of free choline (Cho), PC, and glycerophosphocholine (GPC), displayed a heterogeneous spatial distribution in the tumor. MS imaging performed on tumor sections detected the spatial distributions of individual PC, Cho, and GPC, as well as sodium (Na+) and potassium (K+), among many others. PC and Cho intensity were increased in viable compared with necrotic regions of MDA-MB-231 tumors, but relatively homogeneously distributed in MCF-7 tumors. Such behavior may be related to the role of PC and PC-related enzymes, such as choline kinase, choline transporters, and others, in malignant tumor growth. Na+ and K+ colocalized in the necrotic tumor areas of MDA-MB-231 tumors, whereas in MCF-7 tumors, Na+ was detected in necrotic and K+ in viable tumor regions. This may be attributed to differential Na+/K+ pump functions and K+ channel expressions. Principal component analysis of the MS imaging data clearly identified different tumor microenvironmental regions by their distinct molecular signatures. This molecular information allowed us to differentiate between distinct tumor regions and tumor types, which may, in the future, prove clinically useful in the pathologic assessment of breast cancers.

Monitoring phospholipids for assessment of ion enhancement and ion suppression in ESI and APCI LC/MS/MS for chlorpheniramine in human plasma and the importance of multiple source matrix effect evaluations.

Biological matrix effects are a source of significant errors in both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) LC/MS. Glycerophosphocholines (GPChos) and 2-lyso-glycerophosphocholines (2-lyso GPChos) are known to fragment to form ions at m/z 184 and m/z 104, respectively. Phospholipids were used as markers to evaluate matrix effects resulting in both ion suppression and enhancement using ESI and APCI modes in the determination of chlorpheniramine in human plasma. Results revealed that GPChos and 2-lyso GPChos demonstrated very low ionization efficiency in the APCI mode, post-column infusion experiments were performed to confirm that suppression and enhancement matrix ionization effects coincided with the elution profiles of the phospholipids. The mean matrix effect for chlorpheniramine using APCI was 75% less than the mean matrix effect in ESI, making APCI the ionization method of choice initially even though the absolute response was lower than in the ESI mode. The resulting APCI method showed acceptable results according to the FDA guidelines; however, a multiple source relative matrix effects study demonstrated variability. It was concluded that an absolute matrix effects study in one source of biological fluid may be not sufficient to ensure the validity of the method in various sources of matrix. In order to obviate the multiple matrix source variability, we employed an isotopically labeled internal standard for quantification of chlorpheniramine in the ESI mode. An additional validation was completed with the use of chlorpheniramine-d(6) as the internal standard. This method met all acceptance criteria according to the FDA guidelines, and the relative matrix affects study was successful.